Decreased AdipoR1 signaling and its implications for obesity-induced male infertility

Obesity is among the risk factors for male infertility. Although several mechanisms underlying obesity-induced male subfertility have been reported, the entire mechanism of obesity-induced male infertility still remains unclear. Here, we show that sperm count, sperm motility and sperm fertilizing ability were decreased in male mice fed a high-fat diet and that the expression of the AdipoR1 gene and protein was decreased, and the expression of pro-apoptotic genes and protein increased, in the testis from mice fed a high-fat diet. Moreover, we demonstrate that testes weight, sperm count, sperm motility and sperm fertilizing ability were significantly decreased in AdipoR1 knockout mice compared to those in wild-type mice; furthermore, the phosphorylation of AMPK was decreased, and the expression of pro-apoptotic genes and proteins, caspase-6 activity and pathologically apoptotic seminiferous tubules were increased, in the testis from AdipoR1 knockout mice. Furthermore, study findings show that orally administrated AdipoRon decreased caspase-6 activity and apoptotic seminiferous tubules in the testis, thus ameliorating sperm motility in male mice fed a high-fat diet. This was the first study to demonstrate that decreased AdipoR1/AMPK signaling led to increased caspase-6 activity/increased apoptosis in the testis thus likely accounting for male infertility.


Decreased expression of the AdipoR1, increased expression of pro-apoptotic genes and proteins, and increased TUNEL-positive seminiferous tubules in the testis from mice fed a high-fat diet
To clarify the molecular mechanism by which high-fat diet affects male infertility, we performed gene expression and western blot analyses.High-fat diet significantly decreased the expression of AdipoR1 in the testis compared to that in normal chow diet both in the mRNA and the protein levels (Fig. 2a,b) although there was no significant difference in the expression of AdipoR2 (Adipor2) (Fig. 2c).Moreover, high-fat diet significantly increased the expression of the pro-apoptotic genes, such as Bcl-2-associated X protein (Bax) (Fig. 2d) and caspase-9 (Casp9) (Fig. 2e), as well as the expression of caspase-9 at the protein level (Fig. 2f).Furthermore, we went on to evaluate apoptosis in the testis by terminal transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining and demonstrated that high-fat diet increased the number of apoptotic seminiferous tubules (Fig. 2g,h).

Impaired spermatogenesis and infertility in AdipoR1 KO mice
Based on evidence of decreased expression of the AdipoR1 gene in the testis from mice fed a high-fat diet, we next examined male AdipoR1 KO mice for fertility, and demonstrated that, interestingly, testes weight was significantly decreased (Fig. 3a) and seminiferous tubules were atrophied (Fig. 3b) in AdipoR1 KO mice compared to those in wild-type mice.Moreover, sperm count (Fig. 3c), sperm motility (Fig. 3d), and pregnancy rate (Fig. 3e) were significantly decreased in AdipoR1 KO mice compared to those in wild-type mice.

Increased expression of pro-apoptotic genes and proteins in the testis from AdipoR1 KO mice
We next performed gene expression and western blot analyses on the testis from AdipoR1 KO mice and demonstrated that the expression of pro-apoptotic genes and proteins, such as caspase-9 (Fig. 4a,d), caspase-3 (Fig. 4b,d) and caspase-6 (Fig. 4c,d) were significantly increased in the testis from AdipoR1 KO mice compared to that in wild-type mice.

Decreased phosphorylation of AMPK and increased apoptosis in the testis from AdipoR1 KO mice
Given the report that AdipoR1 activates AMP-activated protein kinase (AMPK) pathways in the liver 28 , we next focused attention on AMPK and demonstrated that the phosphorylation of AMPK was significantly suppressed in the testis from AdipoR1 KO mice compared to that in wild-type mice (Fig. 5a).Then, in light of a recent important finding that AMPK deficiency increases caspase-6 activation in the liver in nonalcoholic steatohepatitis model mice 41 , we hypothesized that the suppression of AMPK activation might induce caspase-6 activation in the testis from AdipoR1 KO mice and investigated caspase-6 activity in the testis from AdipoR1 KO mice.Interestingly, caspase-6 activity was significantly increased in the testis from AdipoR1 KO mice compared to that in wild-type mice (Fig. 5b).
Furthermore, we went on to evaluate apoptosis in the testis by TUNEL staining and demonstrated that the knockout of AdipoR1 increased the number of apoptotic seminiferous tubules (Fig. 5c,d).

Adiponectin/AdipoR1 signaling decreased caspase-6 activity in TM4 cells
AdipoR1 KO mice present with hyperglycemia and insulin resistance 28 , thus suggesting the possibility that hyperglycemia and insulin resistance might contribute to increased apoptosis in the testis from the AdipoR1 KO mice.Then, we studied adiponectin/AdipoR1 signaling in vitro to investigate its direct effect.In TM4 cells incubated with 30 μg/ml adiponectin and 2 mM acadesine (AICAR), caspase-6 activity was significantly decreased (Fig. 6a).Under suppression of AdipoR1 expression with a specific short interfering RNA (siRNA) (Fig. 6b), however, no significant difference was noted in caspase-6 activity between TM4 cells incubated or unincubated with adiponectin (Fig. 6c).These experiments combined to show that adiponectin significantly reduced caspase-6 activity via AdipoR1.

AdipoRon improved sperm motility, and decreased caspase-6 activity and TUNEL-positive seminiferous tubules in the testis from mice fed a high-fat diet
Our research group previously showed that the orally active AdipoR agonist AdipoRon activates AMPK and PPAR-α pathways 22,29 , ameliorates insulin resistance and glucose intolerance 22,29 , and reverses life shortening in obese diabetic mice 29 .Based on evidence that adiponectin/AdipoR1 signaling decreases caspase-6 activity in TM4 cells, we next studied the effects of AdipoRon on sperm parameters and apoptosis in the testis from mice fed a high-fat diet.Interestingly, oral administration of AdipoRon (50 mg per kg body weight) for 14 days had no effect on testes weight (Fig. 7a), however, AdipoRon improved sperm motility (Fig. 7b), activated AMPK in   the testis (Fig. 7c), and decreased caspase-6 activity in the testis (Fig. 7d) and TUNEL-positive seminiferous tubules (Fig. 7e,f).

Discussion
In this study we showed that decreased adiponectin/AdipoR1 signaling is implicated in obesity-induced male infertility and demonstrated for the first time that decreased adiponectin/AdipoR1 signaling involves an increase in caspase-6 activity via the AMPK-caspase-6 axis, thus resulting in suppressed spermatogenesis.AMPK directly phosphorylates caspase-6 to inhibit its cleavage and activation 41 , thus suppressing apoptosis 41 .Caspase-6 has been suggested as an important target in Alzheimer's disease 42,43 as well as in nonalcoholic steatohepatitis 41 , both of which are characterized by decreased AMPK activity 41,44 .Indeed, it is shown that www.nature.com/scientificreports/caspase-6 mediates a feedforward loop to sustain the caspase cascade, where increased activity of upstream executioner caspases, such as caspase-9, -3 and -7, activate the downstream executioner caspase-6, and activated caspase-6 cleaves BH3 interacting-domain death agonist (Bid) and increases mitochondrial cytochrome c release, and then increased cytochrome c release activates caspase-9 in the intrinsic pathway 41 .
The activity of AMPK is regulated by multiple factors, such as nutrients, exercise, hormones and cytokines in physiological or pathological conditions 45,46 .Our research group previously demonstrated that adiponectin/ AdipoR1 signaling activates AMPK in the liver 20,25,28 and the skeletal muscle 30 .AdipoR1 is relatively ubiquitously expressed throughout the body but expressed abundantly in the skeletal muscle, whereas AdipoR2 is found to be predominantly expressed in the liver 46 .Moreover, our research group has shown that adiponectin/AdipoR signaling is decreased in obesity, and decreased adiponectin/AdipoR signaling accounts in part for metabolic syndrome or type 2 diabetes 28,29 .In this study, we showed that testicular adiponectin/AdipoR1 signaling was decreased in obesity.Moreover, we demonstrated that decreased AMPK activity and increased caspase-6 activity in the testis from AdipoR1 KO mice and that AdipoR1 KO mice were associated with male infertility due to smaller testis, lower sperm counts and lower sperm motility.Our study showed that decreased adiponectin/ AdipoR1 signaling accounts for male infertility as it involves apoptosis via the AMPK-caspase-6 axis.
Several reports have shown protective effects of adiponectin on the testis in diabetes model mice 39,40 .In streptozotocin-induced diabetic mice, it was shown that sperm parameters and the protein expression of autophagy were decreased, and the testicular endoplasmic reticulum stress and oxidative stress increased, while recombinant adiponectin treatment reversed these changes 40 .Another research group reported that administration of adiponectin elevated serum testosterone, the expression of testicular steroidogenic marker proteins, insulin receptor, and glucose transporter 8, and intra-testicular concentrations of glucose and lactate and activity of lactate dehydrogenase and antioxidant enzymes in high-fat diet-/streptozotocin-induced diabetic mice, compared to untreated diabetic mice 39 .These findings suggest that adiponectin improves testicular function by increasing the transport of glucose and lactate as well as by reducing oxidative stress.Thus, alongside these, in this study, another mechanism by which adiponectin/AdipoR1 signaling influences testicular functions has been demonstrated for the first time.
Importantly, given that decreased AdipoR expression is a hallmark of obesity 27 , establishing ways to activate adiponectin/AdipoR signal is expected to be the key for definitive treatment of obesity-related diseases.Against this background, small-molecule AdipoR agonists are attracting attention as another potential class of drugs of interest for obesity-related diseases including type 2 diabetes 47,48 .An orally active synthetic small-molecule AdipoR agonist identified by screening a library of candidate compounds 29 , AdipoRon is shown to activate AMPK and PPAR-α pathways 22,29 , ameliorate insulin resistance and glucose intolerance 22,29 , and reverse life shortening  in obese diabetic mice 29 .Moreover, it was recently reported that a PEGylated AdipoRon derivative was more effective in reducing ceramides and dihydroceramides in the liver from mice fed a high-fat diet than AdipoRon 49 .In this study, we demonstrated for the first time that AdipoRon ameliorated sperm motility and apoptosis in the testis from mice fed a high-fat diet.Our data suggested that activating adiponectin/AdipoR1 signaling might be one of the therapeutic targets for obesity-induced male infertility.Given that, based on the structural information of AdipoRs 21 , much progress has been made to date not only in the analysis of the co-crystal structures of AdipoRs and AdipoRon but in the development of small-molecule AdipoR agonists for clinical use 22 , expectations are mounting for AdipoR agonists as an effective therapeutics for obesity-induced male infertility.

Mouse studies
All procedures were carried out in accordance with relevant guidelines and regulations as approved by the Animal Care Committees of The University of Tokyo and the Institute of Medical Science, Asahi Life Foundation, and complied with the standards stated in the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health, revised 2011).The study is reported in accordance with ARRIVE guidelines.Male mice were 6-55 weeks of age at the time of the study.They were housed in cages and maintained on a 12 h light-dark cycle with access to chow and water ad libitum.In these experiments, we used normal chow diet consisting of 24.9% (wt/wt) proteins, 4.6% fibers, 7.1% ashes, 49.5% carbohydrates, 4.8% fat and 9.1% water (CE-2, CLEA Japan Inc.) or high-fat diet consisting of 25.5% (wt/wt) protein, 2.9% fibers, 4.0% ashes, 29.4% carbohydrates, 32.0% fat and 6.2% water (High Fat Diet 32, CLEA Japan Inc.).High-fat diet was fed to male mice from 6 weeks old onwards.C57Bl/6 mice were purchased from Charles River Laboratories Japan, Inc. and Japan SLC, Inc. Akita mice were purchased from Japan SLC, Inc.

Generation of AdipoR1 KO mice
Adipor1 −/− mice (C57Bl/6 background) were generated as described previously 28 .All experiments in this study were conducted on male littermates.

Administration of AdipoRon
For administration, AdipoRon (Combi-Blocks, #QV-9395) was prepared in 0.5% methyl cellulose (WAKO, #133-17815).AdipoRon (50 mg per kg body weight) or 0.5% methylcellulose was orally administrated to high-fat diet-fed mice from 8 weeks of age once daily for two weeks.The sampling of the mice parameters was performed at 10 weeks of age.

Mating assay
Male C57Bl/6 mice fed a high-fat diet for 28 weeks, AdipoR1 KO mice fed standard chow diet, Akita mice fed standard chow diet, and age matched control mice fed standard chow diet were used in a mating assay.Each male mouse was caged with three or four female C57Bl/6 mice for consecutive five days.The percentage of mice achieving a pregnancy were calculated for each male.

Testis and semen analysis in mice
Testis and epididymides were carefully dissected.Bilateral testes were weighed and frozen in liquid nitrogen, and then processed for real-time PCR, western blot analysis or caspase-6 activity assay.Separated cauda epididymis from each mouse was immediately placed into modified HTF medium with HEPES (KITAZATO, #93421) at room temperature.The sperm number in the suspension was counted using a hemocytometer on a Nikon DIAPHOT 300 microscope at 200× magnification.At least 200 sperm were counted in each sperm sample.We assessed motility rate as the percentage of the sum of sperms with progressive motility and non-progressive motility per total sperm number.

Histology and TUNEL staining
For histological examination, each whole testis was fixed in Super Fix (Kurabo, #KY-500) at 4 °C overnight, washed and then was paraffin-embedded and sectioned.Sections were stained with hematoxylin and eosin, and In Situ Cell Death Detection Kit, POD (Roche, #11684817910) following the provided protocol for apoptosis detection.Six to eight micrographs from all regions of the testis were captured for analysis.Seminiferous tubules were evaluated by a trained pathologist for morphometry.At least 90 tubules from all testicular regions were counted for each sample to quantify its apoptotic seminiferous tubules.

Studies with TM4 cells
The mouse Sertoli cell-line TM4 was purchased from American Type Culture Collection (ATCC, #CRL-1715).TM4 cells were cultured in a 1:1 mixture of Ham'S F12 medium and Dulbecco's modified Eagle's medium with 1.

RNA interference
Silencer Select Pre-Designed siRNA (Thermo Fisher Scientific) for Adipor1 (s91210) or Silencer Select Negative Control No.1 siRNA (#4390843) were transfected into TM4 cells by using Lipofectamine RNAiMAX Transfection Reagent.Forty-two hours after transfection, TM4 cells were incubated with D-MEM/Ham's F-12 without horse serum, FBS or penicillin/streptomycin for 10 h, then treated with 30 μg/ml adiponectin for 10 min, or PBS for 10 min, and then the cells were lysed for caspase-6 activity assay.Transfection efficacy was monitored based on gene expression levels of Adipor1 using the real-time PCR.The siRNA sequence for downregulating AdipoR1 is shown in Table S2.

Statistical analysis
Results are expressed as mean ± s.e.m.Differences between two groups were assessed for significance using the unpaired two-tailed t-test.Data involving more than two groups were assessed by analysis of variance (ANOVA) followed by the Dunnett's multiple comparison test or Tukey's honestly significant difference test.Representative data from one of 2-5 independent experiments are shown.Every experiment was performed several times with essentially the same results.

Figure 2 .Figure 3 .Figure 4 .
Figure 2. Expression of AdipoR1 was decreased and expression of pro-apoptotic genes, proteins and TUNELpositive seminiferous tubules were increased in the testis from mice fed a high-fat diet.Adipor1 (a), Adipor2 (c), Bax (d) and Casp9 (e) mRNA levels, and AdipoR1 (b) and caspase-9 (f) protein levels in the testis from mice fed a normal chow diet (NC) and a high-fat diet (HF) for 22-23 weeks.Results of real-time PCR were normalized to Rn18s.Results of western blot were normalized to α-tubulin.Representative micrographs from TUNEL staining of the testis from mice fed a NC and HF (g) and percentage of seminiferous tubules with TUNEL-positive cells in mice fed a NC and HF (h).All values are presented as means ± s.e.m. *P < 0.05 and **P < 0.01 compared to mice fed a NC.P values were determined by the unpaired two-tailed t-test.Scale bar, 100 μm.The red arrows indicate TUNEL-positive cells.NS, not significant.NC, n = 15 (a, c-e), n = 5 (b, h), n = 6 (f); HF, n = 16 (a, c-e), n = 5 (b, h), n = 6 (f).

Figure 5 .
Figure 5. AMPK activity was decreased, and caspase-6 activity and TUNEL-positive seminiferous tubules increased, in the testis from AdipoR1 KO mice.Phosphorylation and amount of AMPK (a), and caspase-6 activity (b) in the testis from wild-type (WT) mice and AdipoR1 knockout (KO) mice.Representative micrographs from TUNEL staining of the testis from WT mice and AdipoR1 KO mice (c) and percentage of seminiferous tubules with TUNEL-positive cells in WT mice and AdipoR1 KO mice (d).Phosphorylation of AMPK were normalized to amount of AMPK.All values are presented as means ± s.e.m. *P < 0.05 and **P < 0.01 compared to WT. P value was determined by the unpaired two-tailed t-test.Scale bar, 100 μm.The red arrows indicate TUNEL-positive cells.WT, n = 6 (a), n = 10 (b), n = 5 (d); AdipoR1 KO, n = 6 (a), n = 10 (b), n = 5 (d).

Figure 7 .
Figure 7. AdipoRon improved sperm motility, and decreased caspase-6 activity and TUNEL-positive seminiferous tubules in the testis from high-fat diet-fed mice.Testes weight (a), sperm motility (b), phosphorylation and amount of AMPK (c), caspase-6 activity in the testis (d), representative micrographs from TUNEL staining of the testis (e), and percentage of seminiferous tubules with TUNEL-positive cells (f), in mice on a high-fat diet, treated once daily with oral administration of AdipoRon (50 mg per kg body weight) for two weeks.Phosphorylation of AMPK were normalized to amount of AMPK.All values are presented as means ± s.e.m. *P < 0.05 and **P < 0.01 compared to control mice.P values were determined by the unpaired two-tailed t-test.NS, not significant.Scale bar, 100 μm.The red arrows indicate TUNEL-positive cells.AdipoRon (−), n = 10 (a, b), n = 5 (c, d, f); AdipoRon (+), n = 10 (a, b), n = 5 (c, d, f).